ABSTRACT Heart failure caused by wild-type transthyretin amyloidosis (ATTRwt) is an underappreciated cause of morbidity and mortality in the aging population. With past support from NIH/NIA (R01AG031804) for studies of ATTRwt, we have characterized the biochemical nature of wild-type TTR in sera and tissues, uncovered a role for clusterin (CLU), identified disease-associated non-coding TTR genetic variants, and accurately defined clinical measures in a large series to identify predictors of survival. Based on these data, we now hypothesize that the pathobiology of ATTRwt involves protein and genetic modifiers, i.e. ATTRwt amyloid fibril formation is related to aberrant or disrupted interactions between TTR and binding partners, clusterin (CLU) and retinol binding protein (RBP4), and the presence of single nucleotide polymorphisms (SNPs) in TTR gene regulatory regions underlie the process. To test our hypothesis, we propose three integrated and translational aims: SA 1. To clarify the amyloidogenic nature of wild-type TTR by interrogating TTR binding partners and SNPs identified in patient samples. Our plan is to a) characterize the structures of CLU and RBP4 isolated from patient and control sera by mass spectrometry (MS), b) define TTR:CLU molecular interactions using MS, surface plasmon resonance, and spectroscopy with attention to CLU glycosylation, stoichiometric effects, and binding site locations, c) study CLU interactions with complexed TTR-RBP4 as in SA1b, and d) correlate TTR SNPs (rs72922940, rs3794885, rs3764479) to CLU and RBP4 structural data from SA1a. SA2. To delineate the roles of TTR SNPs, CLU and RBP4, and the impact of diflunisal (TTR tetramer stabilizing drug) in ATTRwt using a patient-derived, cell-based model. Induced pluripotent stem cells (iPSC), reprogrammed from ATTRwt and control blood monocytes and differentiated into hepatocytes (TTR, CLU, RBP4 expression cell type) or cardiomyocytes (TTR amyloid target cell type) will be used to a) link TTR SNPs to TTR expression/secretion by comparing TTR mRNA half-life/concentrations and protein levels (intracellular and secreted) in ATTRwt hepatocytes +/- SNPs from SA1d; b) define molecular associations of TTR, CLU, and RBP4 secreted by ATTRwt and control hepatocytes using MS and PAGE; and c) compare ATTRwt cardiomyocyte response to ATTRwt hepatocyte-secreted TTR and binding partners +/- diflunisal (and +/- CLU peptides from SA1b) by measuring expression of cardiac cell stress markers (MMP9, HO1, Hsp27, p21, cTnT, BNP) implicated in amyloid pathobiology using qPCR and immunoblot analyses. SA3. To examine associations of TTR, CLU, RBP4, and SNPs with progression and survival in ATTRwt, and the impact of diflunisal on disease course. We will a) measure serum levels of TTR, CLU, and RBP4 in patients at baseline and follow-up evaluations by ELISA, b) correlate serological and genetic data to ATTRwt survival risk factors (BNP, UA, RWT and LVEF), and c) quantify the effect of diflunisal in an observational, controlled study of ATTRwt by assessing investigational and survival risk factors at baseline and follow-up.